This is a proposal to identify and characterize response pathways in Drosophila cells that are triggered by lack of oxygen (hypoxia). During the previous funding period, Dr. Krasnow has developed a Drosophila model system for studying the development of trachea, an epithelial derived tissue that delivers oxygen to larval cells. He has found that there are two cues that dictate the final pattern of the tracheal structure, which resembles a tree with a trunk and branches. First is the developmental genetic program that specifies the stereotypical primary and secondary branch patterns, and the second cue is lack of oxygen that influences the density of terminal branches. It is this second step that is the main focus of this proposal. During the past funding period, Dr. Krasnow has made a number of important discoveries regarding how cells deprived of oxygen produce signals to attract the terminal branches of trachea. The key signal appears to be the Drosophila homolog of Fibroblast Growth Factor (FGF), called Branchless. The synthesis of FGF increases in cells deprived of oxygen. The rise in FGF levels in turn attracts terminal tracheal branches which express the FGF receptor (named Breathless). He has also isolated Drosophila homologs of the two subunits of Hypoxia Inducible Factor I (tango and similar) as well as a factor (VHL) that regulates HIF-1 protein stability. These are proteins known from the work in mammalian systems to be needed for hypoxic response. In a genetic screen, Dr. Krasnow has also identified a potential response gene, named cropped. He shows that Cropped protein localizes to the nucleus in response to hypoxia. The first aim of this proposal is to determine if the five hypoxic response genes -- tango, similar, dVHL, cropped, branchless -- are members of the same or different pathways. Since they were identified by different means (although they all respond to hypoxia) they don't necessarily have to represent a single and unified response to the lack of oxygen. Cropped and HIF-1 are both transcription factors. Dr. Krasnow will first determine if Cropped transcriptionally regulates HIF-1 or vice versa. This will be done by making mutant clones of one gene and examining the expression of the other. The second question will be to ask if Cropped and HIF-1 induces Branchless FGF, the probable output signal of hypoxic conditions. Mutant clones of cropped or HIF-1 (tango) will be induced and Branchless expression will be determined. The rationale for both of these experiments is that if the lack of function in one gene compromises the expression of another gene, they are members of the same response pathway (and establishes their epistatic relationships). Another plan is to determine the relationship of HIF-1 and Branchless to nitric oxide (NO)-mediated response, another hypoxic response pathway recently described in Pat O'Farrel's lab. The second aim is to carry out comprehensive screens for genes that respond to hypoxia. Microarray chips with 8000 Drosophila cDNAs will be screened with probe cDNAs prepared from different sources (including tissue culture cells, animals of different developmental stages, and different tissues) that have been exposed to hypoxic conditions. Microarray chips will also be used to identify genes regulated by HIF-1 (and Cropped). Three criteria will be used to find genes of interest: first, genes with the same induction profile as the known HIF-1 target; two, genes induced in wildtype animals but not in HIF-1 mutant animals; three, genes induced by the over expression of HIF-1. The last aim is to identify additional hypoxia response genes using a genetic screen. The proposed F1 screen involves generating mitotic clones of mutated chromosome arms in larvae and examining the ability of mutant clones to attract terminal tracheal branches. Mutant clones that fail to attract are presumably unable to respond to hypoxia. A variation of the well established FLP/FRT system that allows examination of clones in living larvae is suggested for this purpose.